Millions of patients receive general anesthesia each year from vigilant highly trained experts, who, in part, manage general anesthetic drug toxicities. The safest anesthetic drugs based on LD50:ED50 ratios are etomidate and alphaxalone. Both act primarily as potent and selective modulators of GABAA receptors, the major inhibitory neurotransmitter-gated ion channels in brain. Combinations of anesthetics that act synergisti- cally on GABAA receptors may also provide greater safety by reducing off-target toxicities. The long-term goal of our research is to define both where and how general anesthetics act to modulate GABAA receptor activity. Our previous research for this project has helped map three structurally distinct sets of transmem- brane inter-subunit binding sites for respectively, etomidate (an imidazole), alphaxalone (a neurosteroid), and the stereoselective hypnotic barbiturate R-mTFD-MPAB, and we have identified mutations in these sites that both mimic anesthetic site occupancy and impede anesthetic binding. We also showed that etomidate, propofol, and pentobarbital all act on GABAA receptors, including mutants, in accord with a simple two-state allosteric co-agonist mechanism. However, new preliminary mechanistic and mutant studies indicate that similar models do not account for alphaxalone actions on GABAA receptors. In addition, the effects of mTFD- MPAB on mutant receptors or combined with etomidate in wild-type receptors indicate complex interactions between different sets of anesthetic sites on GABAA receptors. Our new working hypothesis is that the three site-selective anesthetics etomidate, alphaxalone, and R-mTFD-MPAB distinctly affect functional receptor states and that pairs of these drugs differentially synergize in both GABAA receptors and animals. To test these ideas, we propose using molecular models, receptor mutants, multiple electrophysio- logical approaches, mechanistic analysis, and a novel animal model to evaluate key aspects of the hypothesis. In Aim 1, we will define how the mechanisms of alphaxalone and endogenous neurosteroids THDOC and allopregnanolone in GABAA receptors differs from that for etomidate. Mechanistic studies in wild-type and mutant GABAA receptors will be performed in both oocytes (for allosteric model analysis) and HEK293 cells (for rapid kinetic, state-dependence, and phosphorylation studies) to address multiple possibilities. In Aim 2, we will quantify and compare the effects of combining pairs of the three study drugs in wild-type GABAA receptors and assess and compare the effects of mutations in each set of drug sites on actions of all three anesthetics. These studies will utilize quantitative electrophysiology and our unique model-based ?binary? allosteric shift analyses. In Aim 3, we will establish the pharmacodynamic effects of combining pairs of the site-selective anesthetics in a novel aquatic animal model, zebrafish larvae. Video analysis of spontaneous activity and photomotor responses will be used to establish equi-effective drug conditions as a basis for quantitative comparison of different drug pairs and correlation with findings from Aim 2.